Turnover of endogenous ceramide in cultured normal and Farber fibroblasts.
De novo synthesis and turnover of endogenous ceramide in cultured skin fibroblasts from patients affected with Farber lipogranulomatosis were studied by biosynthetical labeling of cellular sphingolipids with [14C]serine. The cellular uptake of [14C]serine and incorporation into de novo synthesized ceramide was similar in normal and Farber fibroblasts, with a half life of newly synthesized ceramide of 2.7 h in normal and diseased cells. Newly synthesized ceramide was found to be channeled directly into biosynthesis of complex sphingolipids rather than contributing to the pool of accumulated ceramide in Farber fibroblasts. The degradation of ceramide generated by the catabolism of complex sphingolipids in Farber cells was greatly delayed compared with control fibroblasts, with differences in the amount of radiolabeled cellular ceramide becoming evident after 6 h chase time. Individual Farber cell lines differed from each other in the amount of accumulated ceramide; however, no correlation was found between ceramide accumulation and residual acid ceramidase activity as determined in vitro. In addition, the amount of radiolabeled sphingomyelin was significantly increased in Farber fibroblasts suggesting a delayed degradation of this compound in this ceramide storage disorder. We propose biosynthetical labeling of endogenous ceramide with [14C]serine, in addition to other established methods, as a highly sensitive and reliable method for the diagnosis of Farber disease, allowing semiquantitative measurement of ceramide accumulation in cultured skin fibroblasts of patients affected with Farber lipogranulomatosis.
van Echten-Deckert
G
G
Kekulé-Institut für Organische Chemie und Biochemie der Universität Bonn, Germany.
Klein
A
A
Linke
T
T
Heinemann
T
T
Weisgerber
J
J
Sandhoff
K
K
D055573:Acid Ceramidase; D000581:Amidohydrolases / Q000378:metabolism; D002250:Carbon Radioisotopes; D002460:Cell Line; D002478:Cells, Cultured; D055572:Ceramidases; D002518:Ceramides / Q000032:analysis / Q000096:biosynthesis / Q000378:metabolism*; D002855:Chromatography, Thin Layer; D005260:Female; D005347:Fibroblasts; D005963:Glucosylceramides / Q000032:analysis / Q000378:metabolism; D006801:Humans; D016464:Lysosomal Storage Diseases / Q000097:blood*; D008297:Male; D010641:Phenotype; D012694:Serine / Q000378:metabolism; D013107:Sphingolipids / Q000096:biosynthesis; D013109:Sphingomyelins / Q000378:metabolism; D013997:Time Factors
D002250:Carbon Radioisotopes; D002518:Ceramides; D005963:Glucosylceramides; D013107:Sphingolipids; D013109:Sphingomyelins; C109343:dihydroceramide; D012694:Serine; C035785:N-lauroylsphingosine; D000581:Amidohydrolases; C526693:ASAH1 protein, human; D055573:Acid Ceramidase; D055572:Ceramidases